Parkinson's disease (PD) is a devastating neurodegenerative disease characterized by motor and non-motor symptoms. The hallmark pathology of PD in the brain is the presence of alpha-synuclein (?-syn) aggregates, along with the loss of dopaminergic neurons in the substantia nigra. The brain ?-syn pathology is thought to start in the gastrointestinal (GI) tract, since both GI dysfunction and the presence of ?-syn aggregates in the GI tract usually precede motor symptoms by many years. In addition, experimental models show that ?-syn aggregates can reach the brain from the gut via the vagal nerve. To understand GI tract contributions to PD, we performed a study that identified the human appendix as a key GI tissue that impacts the risk for PD. This study demonstrated that ?-syn aggregates are abundant in the appendix and that removal of the appendix was associated with a reduced risk of PD. It also showed that the appendix contains aberrant truncated forms of ?-syn, analogous to those in the PD brain, and that these were more abundant in the appendix of PD patients than in healthy individuals. This innovative work provides the basis for a unique opportunity to understand how the appendix contributes to PD. The proposed study will determine how the appendix, and the ?-syn aggregates within, can impact the development of PD. Specifically, this project aims to establish: 1) what gene regulatory changes are prominent in the PD appendix compared to that of healthy controls; 2) the specific truncated forms of ?-syn enriched in the PD appendix and their capacity to seed further aggregation; 3) the consequences of initiating ?-syn pathology in the appendix on the subsequent development of PD-like pathology in the brain, in vivo. This study will generate detailed genome-wide maps of epigenetic abnormalities in the PD appendix ? a resource for understanding gene regulatory and biological pathway changes in the PD GI tract. Profiling the epigenetic mark DNA methylation will be accompanied by an integrative network analysis with transcriptomic data to determine key genes dysregulated in the PD appendix. Next, the identification of the truncated ?-syn proteoforms elevated in the PD appendix, will be carried out using top-down mass spectrometry, the gold-standard for intact protein identification. Furthermore, seeding activity for pathogenic ?-syn in the appendix of PD cases, will be determined and compared to controls, using powerful biochemical assays. Finally, the capacity for ?-syn pathology triggered in the mouse cecal patch ? aka the appendix ? to lead to the development of PD-like neuropathology in the brain, will be determined. We will also determine if intestinal inflammation in combination with ?-syn pathology in the cecal patch augments brain pathology relevant to PD. Together, this study will provide new insights on the mechanisms underlying ?-syn abnormalities in the PD gut and its capacity to induce brain neuropathology. Moreover, this work can help shape the development of early PD diagnostics and treatments directed to the accessible GI tract.